High-resolution structure determination of proteins is critical in understanding the molecular mechanisms of cellular function. The greatest occurrences within genetic diseases are mutations that alter the structure/function of proteins. A major obstacle for obtaining the high-resolution structure of wild type and mutant proteins is the difficulty in expressing proteins of interest in either a homologous or heterologous system at levels needed for structural studies. The current methods for expressing soluble, and especially membrane, proteins rely on fortuity as much as rational design. The inability to over express proteins can be enigmatic and can be a major block for structure determination. The cystic fibrosis transmembrane conductance regulator (CFTR) is a prime example of a critical protein whose structural analysis has been restricted by the inability to express large amounts of the protein. This project will test a novel hypothesis, which directly addresses the cause for low expression of proteins. CFTR is an ideal protein to use to test this hypothesis most importantly because CFTR is predicted by the hypothesis to be difficult to express at high levels. Thus, expression of CFTR at high levels and controls, which reduced expression of CFTR, would be critical tests for the hypothesis. In addition, CFTR is critical and mutations within it are responsible for a major worldwide disease. Overall, if successful, the results of this project will have a high impact on the biomedical field including research on cystic fibrosis. PUBLIC HEALTH RELEVANCE: Rational drug design based on the high resolution structure of novel and known proteins requires systems for high level expression of the proteins. The high level expression of functional protein is a limiting factor for analysis of most membrane proteins, including the protein defective in cystic fibrosis, CFTR. This project will test a novel hypothesis, which if correct will provide a rational approach for successful expression of multidomain proteins with CFTR being used as the test case.